Summary ? Project 1 Merkel cell carcinoma (MCC) is a highly aggressive skin cancer diagnosed in >2,500 persons per year in the US. ~80% of MCCs are caused by the Merkel cell polyomavirus (MCPyV), and these tumors rely on persistently-expressed cell cycle promoting oncoproteins (T-Antigens). These viral T antigens are ideal targets for immunotherapies: they are highly expressed, not readily lost, exclusive to tumor tissue, non-human in sequence (reducing off-target risk), and highly immunogenic. Recent studies have shown that reducing dysfunction of endogenous T cells in MCC is effective, as half of patients have durable responses to PD-1 axis blockade. However, many patients do not respond, representing an unmet clinical need. We hypothesize that patients whose tumors do not respond to PD-1 blockade have insufficient or poorly avid MCPyV-responsive T cells, and that therapeutic efficacy can be improved with a combination of approaches. In an R01-funded trial, we have used ?Triple Therapy? to treat MCC patients with metastatic disease, where we combined single-fraction radiation to reverse MCC-specific downregulation of MHC class I, infusion of autologous ex vivo-expanded MCPyV specific CD8+ T cells to supplement the lack of effective MCPyV-specific T cells and avelumab (a PD-L1 checkpoint inhibitor) to boost antigen-specific responses. These treatments have been well tolerated, encouragingly effective (with 3 of 6 patients achieving complete remission, 2 durable at >2 years), and we have demonstrated persistence, function, and tumor localization of infused T cells. However, this strategy was limited by the rarity and low avidity of the endogenous MCPyV-specific T cells in most patients, and the 2-3 months required to generate T cell products in the face of rapidly progressive disease. To circumvent the challenges encountered, we now propose a transgenic T cell therapy approach. We will 1) use a newly developed high-throughput strategy to identify safe, high-affinity HLA-restricted T cell receptors (TCRs) to ensure all patients can receive highly avid, effective MCPyV-specific T cells. We have successfully used this approach to identify an MCPyV-specific HLA A*0201-restricted TCR (TCRA2-MCC1) and will now employ these strategies to identify TCRs of 3 additional specificities. 2) in a Phase I/II trial for patients with PD- 1 blockade-refractory metastatic MCC, evaluate the safety and efficacy of autologous CD8+ T cells transduced to express the validated TCRA2-MCC1 combined with MHC upregulation and PD-1 axis blockade, and 3) use a suite of cutting-edge tools on patient samples to characterize infused T cells, MCC cells and other features of the tumor microenvironment, identify parameters associated with treatment responses and/or failures. We believe the proposed studies will provide critical insights into the design of next-generation T cell therapies for MCC patients, with implications for other immunogenic malignancies.